Abstract
Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes—while playing an essential role in fern colonization and reproduction—generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.
Highlights
The development of universal DNA barcoding markers for land plants is challenging and the exact choice of loci has been heavily debated [1,2,3]
The Plant Working Group of the Consortium for Barcoding of Life decided on a standard two-locus barcode for all land plants, consisting of portions of the rbcL and matK plastid genes [4]
It was immediately emphasized that this core barcode might have to be augmented with supplementary loci in some groups due to lack of discriminatory power and/or primer universality
Summary
The development of universal DNA barcoding markers for land plants is challenging and the exact choice of loci has been heavily debated [1,2,3]. The Plant Working Group of the Consortium for Barcoding of Life decided on a standard two-locus barcode for all land plants, consisting of portions of the rbcL and matK plastid genes [4]. It was immediately emphasized that this core barcode might have to be augmented with supplementary loci in some groups due to lack of discriminatory power and/or primer universality. None of the currently existing primer sets are likely suitable for all lineages of land plants [10,11] and efforts are focusing on the development of complex primer assays to achieve reliable amplification and sequencing of matK among land plants
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